Hematopoietic stem/progenitor cell (HSPC) communication with the cellular microenvironment is critical for the regulation of stem cell functions. Despite their essential role in the clinical setting, our current understanding of the molecular cues that regulate the trafficking of HSPCs and their niche interactions remain rudimentary. Therefore, to improve HSPC transplantation as a treatment option for patients, it is critical that we identify the molecules and mechanisms that regulate HSPC adhesion, trafficking and repopulation. The objective of this proposal is to determine how the molecular scaffold protein, CD82, regulates HSPC localization and adhesive interactions with the bone marrow, which directly influences HSPC function. We will test the hypothesis that CD82 modulates HSPC homing, mobilization, and long-term repopulation capacity through the regulation of integrin clustering and niche adhesion. In Specific Aim 1, we will use our experience with super-resolution imaging methods and primary human cells to quantify the molecular distribution of CD82 membrane organization and determine the effects on integrin clustering, cell adhesion and homing. For Specific Aim 2, we will determine how changes in CD82 expression alter bone marrow retention, homing, and long-term repopulation of HSPCs using the CD82KO mice. This contribution is significant because we expect to identify CD82 as a novel therapeutic target to improve HSPC isolations and transplant efficacy in the clinic. Furthermore, the combination of quantitative single molecule and in vivo information that we propose to obtain has not been measured previously and will bring new perspectives to the function of CD82 in HSPC adhesion/signaling. As such, this proposal is innovative because it will apply a combinatorial, experimental approach to the problem of HSPC adhesion, bone marrow trafficking and repopulation. By utilizing mouse models, mutational analysis, super-resolution imaging, and primary patient samples, we will integrate molecular, biochemical and morphological information to obtain a multi-scale understanding of the role of CD82 in regulating HSPC/niche interactions and the a41 integrin.